Volume 24, Issue 12, Pages 1406-1414 (June 2014) Resolution Mediator Chemerin15 Reprograms the Wound Microenvironment to Promote Repair and Reduce Scarring  Jenna L. Cash, Mark D. Bass, Jessica Campbell, Matthew Barnes, Paul Kubes, Paul Martin  Current Biology  Volume 24, Issue 12, Pages 1406-1414 (June 2014) DOI: 10.1016/j.cub.2014.05.006 Copyright © 2014 The Authors Terms and Conditions

Current Biology 2014 24, 1406-1414DOI: (10.1016/j.cub.2014.05.006) Copyright © 2014 The Authors Terms and Conditions

Figure 1 Skin Wound Healing Is Accelerated with C15 Treatment through Direct and Indirect Mechanisms Four 4 mm excisional wounds were made to the dorsal skin of Sv129Ev mice. Vehicle or C15 (100 pg/wound) in 30% Pluronic gel was administered directly into the wound immediately after wounding. (A) Schematic diagrams illustrating the location of skin wounds and measurements derived from histological sections. (B) Immunohistochemical staining during the wound repair time course revealed increases in the number of cells expressing chemerin and ChemR23 after wounding, particularly in granulation tissue. (C) Macroscopic photos of vehicle- and C15-treated wounds 0–14 days after wounding. (D) Wound area relative to initial wound area at days 1, 4, and 7 after wounding. (E) Granulation tissue area at wound midpoints. (F) Scab loss 7 days after wounding. (G) Representative photos of sections from day 4 wound midpoints stained with haematoxylin and eosin. (H) Re-epithelialization quantified by measurement of the length of wound epithelial gaps and tongues on days 1, 4, and 7 after wounding. (I) αSMA myofibroblasts in day 4 wounds. (J) HaCaTs (human keratinocytes) were allowed to migrate for 15 hr to fill in a “scratch wound” in vitro in the presence of C15 (1–1,000 pM) or media control. (K) Murine dermal fibroblasts were suspended in a collagen I and media mixture supplemented with 10–1,000 pM C15 or vehicle (media) control. Gels were allowed to solidify at 37°C, and gel contraction was assessed 24 and 48 hr later. Data are expressed as means ± SEM; there were six to ten mice (D and F) or four to eight wounds (B, E, H, and I) per treatment group or four independent experiments (J and K). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 relative to vehicle-treated controls. See also Figure S1. The following abbreviation is used: FOV, field of view. Current Biology 2014 24, 1406-1414DOI: (10.1016/j.cub.2014.05.006) Copyright © 2014 The Authors Terms and Conditions

Figure 2 C15 Inhibits Early Platelet and Neutrophil Inflammatory Events after Cutaneous Wounding A single 580 ± 39 μm2 focal injury was induced on the surface of a dorsal skin flap with a modified electrocautery device. C15 (100 pg/wound) or vehicle (saline) was administered intradermally immediately after wounding. For sham experiments, mice were prepared for intravital microscopy and imaged identically to injured animals, but no injury was induced. (A) Schematic diagram of the skin preparation with burn injury for multichannel fluorescence spinning-disk confocal microscopy. (B) Schematic showing the wound as viewed with a 4× objective; necrotic cells stained with propidium iodide are shown in red. (C) Low-power (4×) views of necrotic cells (propidium iodide, red) and neutrophil recruitment (blue) to vehicle- or C15-treated wounds 2 hr after injury. The scale bar indicates 150 μm. Dotted lines indicate the wound margin, 150 μm and 500 μm from the wound edge. (D) Quantification of neutrophil recruitment to wounds treated with vehicle or C15. (E) High-power (20x) views of neutrophil (Ly6G, blue) and platelet (CD49b, red) behavior within dermal postcapillary venules 2 hr after wounding. The scale bar indicates 30 μm. The arrow in “Sham” indicates a rolling neutrophil. The large arrow in “Burn + vehicle” indicates a neutrophil without interacting platelets. The small arrow indicates neutrophils with interacting platelets. (F–H) Quantification of neutrophil rolling and adhesion (F), platelet rolling and adhesion (G), and platelet-neutrophil interactions (H) after wounding. Data are expressed as means ± SEM; four to eight vessels were visualized per time point in each mouse, and there were four to eight animals per treatment group. Vehicle (n = 4), burn (n = 8), C15 (n = 7). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 relative to vehicle-treated mice. See also Movies S1, S2, and S3. The following abbreviation is used: FOV, field of view. Current Biology 2014 24, 1406-1414DOI: (10.1016/j.cub.2014.05.006) Copyright © 2014 The Authors Terms and Conditions

Figure 3 C15 Dampens Wound Leukocyte Recruitment and Skews Macrophage Phenotype Four 4 mm excisional wounds were made to the dorsal skin of Sv129Ev mice. Vehicle or C15 (100 pg/wound) in 30% Pluronic gel was administered directly into the wound immediately after wounding. (A–C) Representative micrographs and quantification of neutrophil (Ly6G+ cell, brown) (A), mast cell (toluidine blue+ cell, blue) (B), and monocyte-macrophage (F4/80+ cell, brown) (C) recruitment to wound granulation tissue up to 14 days after wounding. (D) Macrophage immunostaining through the midpoint of day 7 wounds. Red arrows indicate wound edges, and green lines mark the edge of the macrophage-dense area. (E) Quantification of the distance that macrophages extend beyond the wound margin into the surrounding tissue. (F) Schematic showing macrophage distribution around vehicle- and C15-treated day 7 wounds and corresponding photos of macrophage morphology. (G) Quantification and representative photos of Prussian blue+ iron-loaded cells within the wound. (H) Quantification and representative photos of TNF-α expression (brown). (I) Characterization of macrophage phenotype within day 7 C15- and vehicle-treated wounds. Ym1 and Arg1 are M2 markers, and iNOS is an M1 macrophage marker. Data are expressed as means ± SEM; there were four to eight wounds per treatment group. ∗p < 0.05 and ∗∗p < 0.01 relative to vehicle-treated wounds. The following abbreviation is used: FOV, field of view. Current Biology 2014 24, 1406-1414DOI: (10.1016/j.cub.2014.05.006) Copyright © 2014 The Authors Terms and Conditions

Figure 4 C15 Inhibits Scar Formation (A) Day 7 wound midsections (4× objective on top; 20× objective on bottom) stained with picrosirius red (collagen) from unwounded skin and vehicle- and C15-treated wounds. (B) Images used for algorithm analysis (zoomed-in areas are shown underneath). Red lines were fitted by the algorithm along white lines of collagen, and the angles of each line were measured and compared for determining fiber alignment. (C) Quantification of collagen fiber alignment in day 7 and 14 wounds. An entirely randomly oriented network of collagen fibers would score 0.5 in this analysis, whereas complete fiber alignment would score 1. Data are expressed as means ± SEM; there were six wounds per treatment group. ∗∗∗p < 0.001 and ∗∗p < 0.01 relative to vehicle-treated wounds. See also Figure S3 and Supplemental Experimental Procedures. Current Biology 2014 24, 1406-1414DOI: (10.1016/j.cub.2014.05.006) Copyright © 2014 The Authors Terms and Conditions